Education

Research summary

The Benner group has:

Initiated synthetic biology as a field. The Benner group was the first to synthesize a gene for an enzyme, and used organic synthesis to prepare the first artificial genetic systems. These systems have been used to direct the synthesis of artificial proteins having unnatural amino acids, in FDA-approved clinical assays for HIV, hepatitis B and hepatitis C that improves the medical care of over 400,000 patients annually, and to support the first artificial chemical system capable of Darwinian evolution.

Invented dynamic combinatorial chemistry, combining ideas from molecular evolution, enzymology, analytical chemistry, and organic chemistry to generate a strategy to discover small molecule therapeutic leads. A German company, Alantos, is today using this technology to develop drug leads.

Established paleomolecular biology, where researchers resurrect ancestral proteins from extinct organisms for study in the laboratory, The strategy allows scientists to connect chemistry to function in biology, which is defined by an organism's fitness in a complex and changing environment.

Helped found evolutionary bioinformatics, in 1991, launched one of the first web-based bioinformatics servers with Gaston Gonnet, generated the first naturally organized protein sequence databases, and helped develop the MasterCatalog that generated ca. $4 million in sales. This work also supported the first exhaustive matching of a modern protein sequence database, the first convincing tools to predict structure in proteins from sequence data, strategies to detect distant homologs using structure prediction, and "post-genomic" tools to detect changing protein function.

Awards

National Science Foundation Graduate Fellow

Junior Fellowship, Harvard Society of Fellows

Dreyfus Award for Young Faculty, 1982

Searle Scholar, 1984-86

Sloan Foundation Fellow, 1984-86

Anniversary Prize, Federation of European Biochemical Societies, 1993

Nolan Summer Award, 1998

Arun Gunthikonda Memorial Award, 1998

Townes R. Leigh Commemorative Professor, 1999

B. R. Baker Award, 2001

Sigma Xi Senior Faculty Award 2005

Fellow of the American Association for the Advancement of Science (Biology) 2015

Honoris Causa, University of Croatia, Romania 2016

Fellow of the International Society for the Study of the Origin of Life (ISSOL) 2017

Antibody fragments such as Fabs possess properties
that can enhance protein and RNA crystallization
and therefore can facilitate macromolecular structure
determination. In particular, Fab BL3-6 binds to
an AAACA RNA pentaloop closed by a GC pair with
~100 nM affinity. The Fab and hairpin have served
as a portable module for RNA crystallization. The potential
for general application make it desirable to
adjust the properties of this crystallization module
in a manner that facilitates its use for RNA structure
determination, such as ease of purification, surface
entropy or binding affinity. In this work, we used both
in vitro RNA selection and phage display selection to
alter the epitope and paratope sides of the binding
interface, respectively, for improved binding affinity.
We identified a 5'-GNGACCC-3' consensus motif in
the RNA and S97N mutation in complimentarity determining
region L3 of the Fab that independently impart
about an order of magnitude improvement in affinity,
resulting from new hydrogen bonding interactions.
Using a model RNA, these modifications facilitated
crystallization under a wider range of conditions and
improved diffraction. The improved features of the
Fab-RNA module may facilitate its use as an affinity
tag for RNA purification and imaging and as a chaperone
for RNA crystallography.

Under the "RNA World" hypothesis, an early episode of
natural history on Earth used RNA as the only genetically encoded
molecule to catalyze steps in its metabolism catalysis. This, according
to the hypothesis, included RNA catalysts that used RNA cofactors.
However, the RNA World hypothesis places special demands on
prebiotic chemistry, which must now deliver not only four
ribonucleosides, but also must deliver the "functional" portion of these
RNA cofactors. While some (e.g. methionine) present no particular
challenges, nicotinamide ribose is special. Essential to its role in
biological oxidations and reductions, its glycosidic bond that holds a
positively charged heterocycle is especially unstable with respect to
cleavage. Nevertheless, we are able to report here a prebiotic
synthesis of phosphorylated nicotinamide ribose under conditions that
also conveniently lead to the adenosine phosphate components of
this and other RNA cofactors.

Directed evolution was first applied to diverse libraries of DNA and RNA molecules a
quarter century ago in the hope of gaining technology that would allow the creation of receptors,
ligands, and catalysts on demand. Despite isolated successes, the outputs of this technology have been
somewhat disappointing, perhaps because the four building blocks of standard DNA and RNA have
too little functionality to have versatile binding properties, and offer too little information density
to fold unambiguously. This review covers the recent literature that seeks to create an improved
platform to support laboratory Darwinism, one based on an artificially expanded genetic information
system (AEGIS) that adds independently replicating nucleotide "letters" to the evolving "alphabet".

A common criticism of "prebiotic chemistry research" is that it is done
with starting materials that are too pure, in experiments that are too directed, to get
results that are too scripted, under conditions that could never have existed on Earth.
Planetary scientists in particular remark that these experiments often arise simply
because a chemist has a "cool idea" and then pursues it without considering external
factors, especially geological and planetary context. A growing literature addresses
this criticism and is reviewed here. We assume a model where RNA emerged
spontaneously from a prebiotic environment on early Earth, giving the planet its
first access to Darwinism. This "RNA First Hypothesis" is not driven by the intrinsic
prebiotic accessibility; quite the contrary, RNA is a "prebiotic chemist's nightmare."
However, by assuming models for the accretion of the Earth, the formation of the
Moon, and the acquisition of Earth's "late veneer," a reasonable geological model
can be envisioned to deliver the organic precursors needed to form the nucleobases
and ribose of RNA. A geological model having an environment with dry arid land
under a carbon dioxide atmosphere receiving effluent from serpentinizing igneous
rocks allows their conversion to nucleosides and nucleoside phosphates. Mineral
elements including boron and molybdenum prevent organic material from devolving
to form "tars" along the way. And dehydration and activation allows the formation of
oligomeric RNA that can be stabilized by adsorption on available minerals.

Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°37) for oligonucleotide hybridization were extracted. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair observed for C:O6-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodynamics of oligonucleotides containing Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.

The prebiotic significance of laboratory experiments that study the interactions between oligomeric RNA and mineral species is difficult to know. Natural exemplars of specific minerals can differ widely depending on their provenance. While laboratory-generated samples of synthetic minerals can have controlled compositions, they are often viewed as "unnatural". Here, we show how trends in the interaction of RNA with natural mineral specimens, synthetic mineral specimens, and co-precipitated pairs of synthetic minerals, can make a persuasive case that the observed interactions reflect the composition of the minerals themselves, rather than their being simply examples of large molecules associating nonspecifically with large surfaces. Using this approach, we have discovered Periodic Table trends in the binding of oligomeric RNA to alkaline earth carbonate minerals and alkaline earth sulfate minerals, where those trends are the same when measured in natural and synthetic minerals. They are also validated by comparison of co-precipitated synthetic minerals. We also show differential binding of RNA to polymorphic forms of calcium carbonate, and the stabilization of bound RNA on aragonite. These have relevance to the prebiotic stabilization of RNA, where such carbonate minerals are expected to have been abundant, as they appear to be today on Mars.

Nucleobase pairs in DNA match hydrogen-bond donor and
acceptor groups on the nucleobases. However, these can adopt
more than one tautomeric form, and can consequently pair with
nucleobases other than their canonical complements, possibly
a source of natural mutation. These issues are now being revisited
by synthetic biologists increasing the number of replicable
pairs in DNA by exploiting unnatural hydrogen bonding patterns,
where tautomerism can also create mutation. Here, we combine
spectroscopic measurements on methylated analogs of isoguanine
tautomers and tautomeric mixtures with statistical analyses
to a set of isoguanine analogs, the complement of isocytosine, the
5th and 6th "letters" in DNA.

According to a current "RNA first" model for the origin of life, RNA
emerged in some form on early Earth to become the first biopolymer
to support Darwinism here. Threose nucleic acid (TNA) and
other polyelectrolytes are also considered as the possible first Darwinian
biopolymer(s). This model is being developed by research
pursuing a "Discontinuous Synthesis Model" (DSM) for the formation
of RNA and/or TNA from precursor molecules that might have
been available on early Earth from prebiotic reactions, with the goal
of making the model less discontinuous. In general, this is done by
examining the reactivity of isolated products from proposed steps
that generate those products, with increasing complexity of the reaction
mixtures in the proposed mineralogical environments. Here,
we report that adenine, diaminopurine, and hypoxanthine nucleoside
phosphates and a noncanonical pyrimidine nucleoside (zebularine)
phosphate can be formed from the direct coupling reaction of
cyclic carbohydrate phosphates with the free nucleobases. The reaction
is stereoselective, giving only the β-anomer of the nucleotides
within detectable limits. For purines, the coupling is also
regioselective, giving the N-9 nucleotide for adenine as a major
product. In the DSM, phosphorylated carbohydrates are presumed
to have been available via reactions explored previously [Krishnamurthy
R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed
39:2281-2285], while nucleobases are presumed to have been available
from hydrogen cyanide and other nitrogenous species formed
in Earth's primitive atmosphere.

To the astrobiologist, Enceladus offers easy access to a potential subsurface biosphere via the intermediacy of a
plume of water emerging directly into space. A direct question follows: If we were to collect a sample of this
plume, what in that sample, through its presence or its absence, would suggest the presence and/or absence of
life in this exotic locale? This question is, of course, relevant for life detection in any aqueous lagoon that we
might be able to sample. This manuscript reviews physical chemical constraints that must be met by a genetic
polymer for it to support Darwinism, a process believed to be required for a chemical system to generate
properties that we value in biology. We propose that the most important of these is a repeating backbone charge;
a Darwinian genetic biopolymer must be a "polyelectrolyte". Relevant to mission design, such biopolymers are
especially easy to recover and concentrate from aqueous mixtures for detection, simply by washing the aqueous
mixtures across a polycharged support. Several device architectures are described to ensure that, once captured,
the biopolymer meets two other requirements for Darwinism, homochirality and a small building block "alphabet."
This approach is compared and contrasted with alternative biomolecule detection approaches that seek
homochirality and constrained alphabets in non-encoded biopolymers. This discussion is set within a model
for the history of the terran biosphere, identifying points in that natural history where these alternative approaches
would have failed to detect terran life. Key Words: Enceladus-Life detection-Europa-Icy moon-
Biosignatures-Polyelectrolyte theory of the gene. Astrobiology 17, 840-851.

One frontier in synthetic biology seeks to move artificially
expanded genetic information systems (AEGIS) into natural living cells and to
arrange the metabolism of those cells to allow them to replicate plasmids built
from these unnatural genetic systems. In addition to requiring polymerases that
replicate AEGIS oligonucleotides, such cells require metabolic pathways that
biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those
polymerases. Such pathways generally require nucleoside and nucleotide kinases
to phosphorylate AEGIS nucleosides and nucleotides on the path to these
triphosphates. Thus, constructing such pathways focuses on engineering natural
nucleoside and nucleotide kinases, which often do not accept the unnatural
AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the
enzyme engineer to follow the kinase reaction, assays that are easily confused by
ATPase and other spurious activities that might arise through "site-directed
damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural
and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring
the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-
(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-
4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro
and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

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With cartoons by Jake Fuller, Steven Benner explains how scientists tackle big questions: What is life? How did it begin? If we encounter life in our galactic travels, how would we know it? Learn more.